Phosphonocephem compound

ABSTRACT

A cephem compound (particularly its crystal) represented by the formula [I], wherein X is CH 3 COOH, CH 3 CH 2 COOH or CH 3 CN, and n is 0 to 5, is useful as an antibacterial agent (particularly anti-MRSA agent) and shows superior quality such as high solid stability, possible long-term stable preservation and the like.

This application is the National Phase filing of International PatentApplication No. PCT/JP01/06904, filed 10 Aug. 2001.

TECHNICAL FIELD

The present invention relates to a compound (particularly a crystalthereof) useful as a pharmaceutical agent of a phosphonocephem compoundhaving a superior antibacterial activity and a production methodthereof.

BACKGROUND ART

JP-A-11-255772 discloses phosphonocephem compounds having a superiorantibacterial activity, wherein a lyophilized product of7β-[2(Z)-ethoxyimino-2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)acetamido]-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylaterepresented by the formula:

is described as one of the specific examples thereof.

In general, pharmaceutical agents are desired to be superior in qualitysuch as absorbability, solubility, purity, stability, preservability,tractability and the like. Thus, the problem of the present invention isto provide an antibacterial agent (particularly anti-MRSA agent) havingsuch quality as sufficiently satisfactory as a pharmaceutical product.

DISCLOSURE OF THE INVENTION

The present inventors have intensively conducted various studies in viewof the above-mentioned problem, selected, from among a number ofphosphonocephem compounds, a phosphonocephem compound having aparticular chemical structure represented by the above-mentioned formula(Ia) and found that, when this compound, water and a particular solvent(CH₃COOH, CH₃CH₂COOH or CH₃CN) are mixed and dissolved, a compound(particularly a crystal obtained by successful crystallization) havingan antibacterial activity, which is particularly superior in quality asa pharmaceutical product, can be obtained unexpectedly, and that thiscompound has more than sufficient superior quality as a pharmaceuticalproduct (e.g., having high solid stability and high purity, and thelike), which resulted in the completion of the present invention.

Accordingly, the present invention relates to

-   (1) a compound of the formula:    wherein X is CH₃COOH, CH₃CH₂COOH or CH₃CN, and n is 0 to 5,-   (2) the compound of the above-mentioned (1), which is in the form of    a crystal,-   (3) the compound of the above-mentioned (1), wherein n is 1,-   (4) the compound of the above-mentioned (1) or (2), wherein X is    CH₃COOH,-   (5) the compound of the above-mentioned (4), having peaks near    diffraction angles of 16.32, 19.06, 19.90, 20.98 and 23.24° in    powder X-ray diffraction,-   (6) the compound of the above-mentioned (4), having peaks near    diffraction angles of 11.82, 17.16, 17.80, 19.32, 20.00, 21.20,    21.78, 22.94, 24.10 and 27.02° in powder X-ray diffraction,-   (7) the compound of the above-mentioned (1) or (2), wherein X is    CH₃CH₂COOH,-   (8) the compound of the above-mentioned (7), having peaks near    diffraction angles of 16.30, 18.84, 19.70, 21.80 and 23.18° in    powder X-ray diffraction,-   (9) a pharmaceutical composition, comprising the compound of the    above-mentioned (1) or (2),-   (10) the pharmaceutical composition of the above-mentioned (9),    which is an antibacterial agent,-   (11) a production method of a crystal of a compound represented by    the formula:    wherein X is CH₃COOH, CH₃CH₂COOH or CH₃CN, and n is 0 to 5, which    comprises mixing [i] a compound represented by the formula:    [ii] CH₃COOH, CH₃CH₂COOH or CH₃CN and [iii] water, dissolving them    and allowing crystallization to take place,-   (12) the production method of the above-mentioned (11), wherein the    proportion (volume ratio) to be used of CH₃COOH, CH₃CH₂COOH or    CH₃CN:water is 1:0.1-10,-   (13) a crystal obtained by mixing [i] a compound represented by the    formula:    [ii] CH₃COOH, CH₃CH₂COOH or CH₃CN and [iii] water, dissolving them,    and allowing crystallization to take place,-   (14) a disodium salt of a compound represented by the formula:-   (15) the disodium salt of the above-mentioned (14), which is in the    form of a crystal,-   (16) the disodium salt of the above-mentioned (14) or (15), having a    peak near a diffraction angle of 17.02, 18.94, 22.86, 23.36 or    26.48° in powder X-ray diffraction, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) ofthe compound obtained in Example 1, wherein the transverse axis shows adiffraction angle (2θ) and the vertical axis shows a peak intensity.

FIG. 2 shows a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) ofthe compound obtained in Example 5, wherein the transverse axis shows adiffraction angle (2θ) and the vertical axis shows a peak intensity.

FIG. 3 shows a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) ofthe compound obtained in Example 6, wherein the transverse axis shows adiffraction angle (2θ) and the vertical axis shows a peak intensity.

FIG. 4 shows a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) ofthe compound obtained in Example 7, wherein the transverse axis shows adiffraction angle (2θ) and the vertical axis shows a peak intensity.

FIG. 5 shows a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) ofthe compound obtained in Example 24, wherein the transverse axis shows adiffraction angle (2θ) and the vertical axis shows a peak intensity.

DETAILED DESCRIPTION OF THE INVENTION

The compound (I) consists of 1 molecule of compound (Ia), 1 molecule ofacetic acid, propionic acid or acetonitrile, and 0 to 5 molecules ofwater. The compound (I) may be a salt formed by compound (Ia) and aceticacid or propionic acid, or a solvate formed by compound (Ia) and aceticacid, propionic acid or acetonitrile. The compound (I) may containwater, in which case water may be incorporated as crystal water orsimply adhering water. The compound (I) preferably takes the form of acrystal in view of purity and solid stability.

The compound (I) can be produced from [i] compound (Ia), [ii] aceticacid, propionic acid or acetonitrile and [iii] water. As the compound(Ia), a lyophilized product described in JP-A-11-255772 may be used, orcompound (Ia) obtained by adding, for example, hydrochloric acid,sulfuric acid, nitric acid and the like to a solution containing adisodium salt, dipotassium salt or diammonium salt of compound (Ia). Thesolution containing the above-mentioned disodium salt of compound (Ia)may be a reaction mixture containing a disodium salt of compound (Ia),which is obtained as a result of various reactions for synthesizing adisodium salt of compound (Ia), or may be one wherein a crystal ofdisodium salt of compound (Ia) is dissolved. The compound (I) can beproduced by mixing the above-mentioned [i], [ii] and [iii] to give asolution, and isolating from this solution by a general isolationtechnique such as crystallization and the like. The order of mixing [i],[ii] and [iii] is arbitrary. For example, [i] and [ii] are first mixedand the mixture may be mixed with [iii], or [i] and [iii] are mixed andthe mixture may be mixed with [ii], or [ii] and [iii] are mixed and themixture may be mixed with [i]. To give a solution after mixing, forexample, ultrasonication (ultrasonic irradiation), stirring and the likeare preferably applied. Upon making a solution, crystals may besimultaneously produced. When crystallization does not occur,crystallization may be caused by, for example, cooling, givingstimulation such as ultrasonication, stirring and the like, adding aseed crystal, and the like. To control physical property of theprecipitated crystals, crystallization may be caused after addingsaccharides to the mixture.

The mixing ratio of solvent [ii] and water [iii] for crystallization ina volume ratio of [ii]:[iii] is generally 1:0.1-10, preferably 1:0.5-5,particularly preferably about 1:1. The amounts of [ii] and [iii] to beused relative to [i] for crystallization are not particularly limited aslong as they are within the range that allows for crystallization. Thetotal amount of [ii] and [iii] relative to 1 part by weight of [i] isgenerally 2-100 parts by weight, preferably 3-50 parts by weight, stillpreferably 5-30 parts by weight. The crystallization can be conducted bya crystallization technique such as cooling the above-mentionedsolution, reducing the amounts of [ii] and [iii] in vacuo, adding a seedcrystal and the like. The saccharides can be added to the mixed solutionin an amount up to the maximum amount that can be dissolved in [iii]. Itis added relative to [i] generally in 0.05-10 parts by weight,preferably 0.1-0.5 part by weight. Generally, saccharide is preferablydissolved in [iii] for use. Examples of the saccharide include glucose,mannitol, sucrose, sorbitol, xylitol, fructose, maltose and the like,particularly preferably glucose and mannitol.

The compound (I) thus obtained can be separated from the solution by ageneral separation technique (e.g., filtration, centrifugation and thelike) and purified by a general purification technique (e.g., washingwith solvent and the like).

Because the thus-obtained compound (I) (particularly its crystal) has,for example, high purity and is superior in solid stability and thelike, it can be used as a pharmaceutical preparation.

The crystal of compound (I) can have different water content accordingto the degree of drying. One free of water is encompassed in the scopeof the present invention.

The compound (Ia) can be converted to a dialkali salt such as disodiumsalt, dipotassium salt, diammonium salt and the like. The dialkalinesalt of compound (Ia) can be produced by, for example, adding, forexample, sodium hydroxide, sodium carbonate, sodium acetate, sodiumhydrogencarbonate, potassium hydroxide, potassium carbonate, potassiumacetate, potassium bicarbonate, aqueous ammonia, ammonium carbonate,ammonium acetate and the like to alkalify a solution containing compound(Ia) [e.g., a reaction solution containing compound (Ia) immediatelyafter production (when hydrophilic solvent is not contained, it isadded), a solution of compound (Ia) dissolved in hydrophilic solvent,and the like]. The hydrophilic solvent is, for example, organic acidssuch as acetic acid, propionic acid, lactic acid, succinic acid and thelike, nitrites such as acetonitrile and the like, ketones such asacetone and the like, alcohols such as methanol, ethanol and the like,ethers such as dioxane, tetrahydrofuran and the like, and the like, andhydrophilic organic solvent such as mixed solvents thereof and the like,and a mixed solvent thereof with water. Of these, acetic acid, propionicacid, acetonitrile, methanol, ethanol, lactic acid and a mixed solventthereof with water are preferable, particularly, acetonitrile, methanol,ethanol, acetic acid, propionic acid, and a mixed solvent thereof withwater are preferable.

The crystal of disodium salt of compound (Ia) can be produced by, forexample, crystallization of disodium salt of compound (Ia) from theabove-mentioned hydrophilic solvent.

When crystallization is conducted, the amount of disodium salt ofcompound (Ia) and the solvent to be used is not particularly limited aslong as crystallization can take place. It is generally 2-100 parts byweight, preferably 3-50 parts by weight, still preferably 5-30 parts byweight, of the solvent per part by weight of the disodium salt. Thecrystallization can be conducted by a crystallization technique such ascooling the above-mentioned solution, reducing the amounts of solventand water in vacuo, adding a seed crystal and the like.

The crystal obtained by such crystallization is separated from thesolution by a general separation technique (e.g., filtration,centrifugation and the like) and can be purified by a generalpurification technique (e.g., washing with solvent and the like).

Because the thus-obtained crystal of disodium salt of compound (Ia) hashigh purity, it can be used for producing compound (Ia) and the like.For example, when producing compound (Ia), the compound (Ia) is oncecrystallized as its disodium salt, the crystal is separated anddissolved in the aforementioned hydrophilic solvent, to whichhydrochloric acid, sulfuric acid, nitric acid or the like is added toconvert the compound to compound (Ia), whereby compound (Ia) can beobtained at high purity and in high yield.

Since the compound (I) has a superior antibacterial activity and abroad-spectrum antibacterial activity and shows low toxicity, it can beused safely for the prophylaxis or treatment of various diseases invarious mammals (e.g., mouse, rat, rabbit, dog, cat, cattle, pig and thelike) including human, which are caused by pathogenic bacteria, such assinopulmonary infection and urinary tract infection. The bacteria to bethe target when compound (I) is used as an antibacterial agent are notparticularly limited as long as compound (I) shows an antibacterialactivity thereon, and a wide range of gram-positive bacteria andgram-negative bacteria can be the target. The compound (I) particularlyshows a superior antibacterial activity against staphylococcus andmethicillin-resistant staphylococcus aureus (MRSA). It is consideredthat the compound (I) is converted to a compound represented by theformula:

[hereinafter referred to simply as compound (II)] in biologicalorganisms and shows an antibacterial activity.

The compound (I) (particularly its crystal) shows superior stability andcan be parenterally or orally administered as injection, capsule, tabletor granule, like known penicillin preparations and cephalosporinpreparations, and is preferably administered particularly as aninjection. When it is administered as an injection, the dose thereof ascompound (I) is, for example, generally 0.5-80 mg/day, more preferably2-40 mg/day, per 1 kg of the body weight of a human or animal infectedwith the aforementioned pathogenic bacteria, and is generallyadministered in 2 or 3 times a day in divided doses.

When it is used as an injection, the crystal of compound (I) and asolvent (e.g., distilled water, physiological saline, 5% glucosesolution and the like) are generally packaged separately to provide aninjection, and the crystal of compound (I) is dissolved in a solventwhen in use for administration. It is also possible to administercompound (I) after mixing with a medical infusion solution such asclinical nutrition and the like. It is preferable that the injectiongenerally contain a pH adjuster, examples of which include carbonate,phosphate, acetate and citrate of alkaline metal or alkaline earth metal(e.g., carbonates such as sodium carbonate, potassium carbonate, sodiumhydrogencarbonate, potassium bicarbonate, calcium carbonate and thelike, phosphates such as trisodium phosphate, sodium dihydrogenphosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate,potassium dihydrogen phosphate, calcium hydrogen phosphate, calciumdihydrogen phosphate and the like, acetates such as sodium acetate,potassium acetate, calcium acetate and the like, citrates such asdisodium citrate, sodium citrate, sodium dihydrogen citrate, calciumcitrate and the like), basic amino acids (e.g., L-arginine, L-lysin andthe like), N-methylglucamine and the like, and of these, an injectioncontaining carbonate of alkaline metal or alkaline earth metal and abasic amino acid is preferable. L-Arginine is particularly preferable.Such pH adjusters are used in an amount that makes the pH of theinjection solution when in use 4 to 10, preferably 4.5 to 8.5, morepreferably 5.0 to 8.0, still preferably 5.0 to 7.5. When L-arginine isused, it is used in an amount of generally 0.1 equivalent-5.0equivalents, preferably 2.0-3.5 equivalents, more preferably 2.5-3.2equivalents, relative to the active component. These pH adjusters aregenerally packaged in a state in which they are dissolved in a solvent.However, they may be mixed with the crystal of compound (I) andpackaged, or may be packaged separately from the crystal of compound (I)and the solvent, and mixed when in use. Moreover, the stability of aninjection solution when in use can be enhanced by adding solubilizingagents having reductability to the above-mentioned injection. Examplesof the solubilizing agents having reductability include sodium sulfite,sodium hydrosulfite, sodium hydrogen sulfite, sodium pyrosulfite,L-cysteine and the like. These reducing agents are used in an amount ofgenerally 0.001 equivalent-2.0 equivalents, preferably 0.01-0.5equivalent, more preferably 0.05-0.2 equivalent, relative to the activecomponent. These solubilizing agents having reductability are generallypackaged in a state in which they are divided in a solvent. However,they may be mixed with the crystal of compound (I), may be mixed with apH adjuster, or may be packaged separately from the crystal of compound(I), the pH adjuster and the solvent and mixed when in use.

The content of the crystal of compound (I) in an injection preparation,calculated as compound (Ia), is 100-2000 mg, preferably 200-1000 mg.

The proportion of a solvent in an injection preparation in weight ratiois 10-500, preferably 20-300, relative to the crystal of compound (I)as 1. The proportion of a pH adjuster is generally 1.0-3.0 equivalentsof pH adjuster relative to 1 equivalent of the crystal of compound (I),calculated as compound (Ia).

The pharmaceutical composition of the present invention may contain onlycompound (I), or may contain a carrier generally used for pharmaceuticalagents (e.g., solvent and the above-mentioned pH adjuster in the case ofinjection) and the like.

Of the compounds (I), a compound wherein X is CH₃COOH or CH₃CH₂COOH anda crystal thereof are preferable. When X is CH₃COOH, a compound havingpeaks near diffraction angles of 16.32, 19.06, 19.90, 20.98 and 23.24°in powder X-ray diffraction, and a compound having peaks neardiffraction angles of 11.82, 17.16, 17.80, 19.32, 20.00, 21.20, 21.78,22.94, 24.10 and 27.02° in powder X-ray diffraction are particularlypreferable. When X is CH₃CH₂COOH, a compound having peaks neardiffraction angles of 16.30, 18.84, 19.70, 21.80 and 23.18° in powderX-ray diffraction is particularly preferable. The “near” in theabove-mentioned diffraction angle means ±0.2°.

Now the production methods of compound (Ia) used as a starting materialin the present invention are described.

In the formulas (III), (V) and (VII), Ph shows phenyl group, BH showsbenzhydryl group, in the formula (VIII), BH shows benzhydryl group, inthe formula (IV), M shows alkali metal atom such as lithium, sodium,potassium and the like or alkaline earth metal atom such as magnesium,calcium, barium and the like, and i-BuOH shows isobutanol.

First, compound (III) and a compound of the formula (IV) [hereinafterreferred to simply as compound (IV)] are reacted to give a compound ofthe formula (V) [hereinafter referred to simply as compound (V)]. Thecompound (III) is a known compound, and described in, for example, J.Org. Chem. 1989, 54, 4962-4966. This reaction is generally carried outin the presence of a solvent, and preferably carried out in an inertsolvent such as hydrocarbons (e.g., toluene and the like), esters (e.g.,ethyl acetate and the like), ketones (e.g., acetone and the like),halogenated hydrocarbons (e.g., chloroform, dichloromethane and thelike), ethers (e.g., diethyl ether, tetrahydrofuran, dioxane and thelike), nitrites (e.g., acetonitrile and the like), alcohols (e.g.,methanol, ethanol, n-propanol and the like), amides (e.g.,dimethylformamide, dimethylacetamide and the like), sulfoxides (e.g.,dimethyl sulfoxide and the like), and the like. A mixture of 2 or 3kinds of these solvents may be used as a solvent, and a mixture of theabove-mentioned solvent with water can be also used as a solvent. Ofthese, tetrahydrofuran, acetonitrile, methanol and the like arepreferable, particularly tetrahydrofuran, methanol and a mixture of thetwo are preferable. The amount of compound (IV) to be used is generally1 to 3 mol, preferably 1 to 2 mol, per 1 mol of compound (III). Thereaction temperature is from −40° C. to 80° C., preferably from −20° C.to 50° C. The reaction time is 5 min-12 hr, preferably 30 min-8 hr.Where necessary, a base and a salt can be added to this reaction, whichcan accelerate the reaction. Examples of such base and salts includeinorganic bases such as sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, sodium hydride and the like, alkoxidessuch as sodium methoxide, t-butoxypotassium, t-butoxysodium and thelike, and organic amines such as trialkylamine (e.g., triethylamine,ethyldiisopropylamine and the like). Of these, sodium hydroxide andsodium methoxide are preferable. As the salt, quaternary ammonium saltsuch as tetrabutylammonium salt, and the like are used.

The compound (VII) can be produced by reacting compound (V) and reagentfor forming quaternary ammonium such as iodomethane represented by theformula (VI) [hereinafter referred to simply as compound (VI)] and thelike. This reaction is generally carried out in the presence of asolvent. Examples of the solvent generally include inert solvents suchas hydrocarbons (e.g., toluene and the like), ketones (e.g., acetone andthe like), halogenated hydrocarbons (e.g., chloroform, dichloromethaneand the like), ethers (e.g., diethyl ether, tetrahydrofuran, dioxane andthe like), nitriles (e.g., acetonitrile and the like), alcohols (e.g.,methanol, ethanol, n-propanol and the like), amides (e.g.,dimethylformamide, dimethylacetamide and the like), sulfoxides (e.g.,dimethyl sulfoxide and the like) and the like. A mixture of 2 or 3 kindsof these solvents may be used as a solvent, and a mixture of theabove-mentioned solvent with water can be also used as a solvent. Ofthese, tetrahydrofuran, dimethylformamide, acetonitrile and dimethylsulfoxide are preferable, particularly tetrahydrofuran anddimethylformamide are preferable. The amount of reagent for formingquaternary ammonium to be used is generally 1-20 mol, preferably 1-10mol, per 1 mol of compound (V). The reaction temperature is from −5° C.to 80° C., preferably from 0° C. to 50° C. The reaction time is 30min-48 hr, preferably 2 hr-20 hr.

A compound represented by the formula (VIII) [hereinafter referred tosimply as compound (VIII)] can be produced by subjecting compound (VII)to deprotection of amino group. This reaction is carried out using 1-10mol, preferably 1-5 mol, of phosphorus pentachloride and 1-10 mol,preferably 1-5 mol, of tertiary amine (e.g., pyridine,N,N-dimethylaniline, picoline, lutidine and the like), relative tocompound (VII), which are reacted in an inert solvent and reacting with2-200 mol, preferably 3-125 mol, of alcohols (e.g., methanol, ethanol,isobutanol, isopropyl alcohol and the like). Preferable tertiary amineis exemplified by pyridine and N,N-dimethylaniline, and pyridine isparticularly preferable. Preferable alcohol is exemplified by methanol,ethanol and isobutanol, and methanol and isobutanol are particularlypreferable. Examples of the inert solvent generally include hydrocarbons(e.g., toluene and the like), esters (e.g., ethyl acetate and the like),ketones (e.g., acetone and the like), halogenated hydrocarbons (e.g.,chloroform, dichloromethane and the like), ethers (e.g., diethyl ether,tetrahydrofuran, dioxane and the like), nitriles (e.g., acetonitrile andthe like), amides (e.g., dimethylformamide, dimethylacetamide and thelike), and sulfoxides (e.g., dimethyl sulfoxide and the like). A mixtureof 2 or 3 kinds of these solvents may be used as a solvent. Of these,halogenated hydrocarbons such as chloroform, dichloromethane and thelike are preferable, and dichloromethane is particularly preferable. Thereaction temperature is from −40° C. to 80° C., preferably from −20° C.to 50° C. The reaction time is 30 min-48 hr, preferably 2 hr-24 hr.

Then, compound (VIII) is subjected to deprotection of carboxyl group togive a compound of the formula (IX) [hereinafter referred to simply ascompound (IX)]. This reaction is generally carried out using an acid ina solvent. Examples of the solvent generally include inert solvents suchas hydrocarbons (e.g., toluene and the like), ketones (e.g., acetone andthe like), halogenated hydrocarbons (e.g., chloroform, dichloromethaneand the like), ethers (e.g., diethyl ether, tetrahydrofuran, dioxane andthe like), nitriles (e.g., acetonitrile and the like), esters (e.g.,ethyl acetate and the like), alcohols (e.g., methanol, ethanol,n-propanol and the like), amides (e.g., dimethylformamide,dimethylacetamide and the like), sulfoxides (e.g., dimethyl sulfoxideand the like) and the like. A mixture of 2 or 3 kinds of these solventsmay be used as a solvent. Of these, halogenated hydrocarbons such aschloroform, dichloromethane and the like, nitriles such as acetonitrileand the like, and esters such as ethyl acetate and the like arepreferable, and acetonitrile, ethyl acetate and a mixture of the two areparticularly preferable. As the acid, hydrochloric acid ortrifluoroacetic acid is preferable, and hydrochloric acid isparticularly preferable. The amount of the acid to be used is 2-200 mol,preferably 3-50 mol, relative to compound (VIII). In this case, anisole,phenol and the like are preferably added as a cation scavenger toaccelerate the reaction. The reaction temperature is from −40° C. to 80°C., preferably from −20° C. to 50° C. The reaction time is 30 min-48 hr,preferably 2 hr-24 hr.

The compound (IX) obtained by this production method is generallyobtained as an addition salt with one or two acids, and can be taken outfrom an organic solvent, water or a mixture of the both as a crystal ofan addition salt with one or two acids. As the acid of the addition saltwith one or two acids, mineral acid and organic acid are mentioned. Ofthese, hydrochloric acid, sulfuric acid and trifluoroacetic acid arepreferable, and hydrochloric acid is particularly preferable. Theorganic solvent to be used is exemplified by the aforementioned inertsolvents. Of those, acetonitrile, ethyl acetate, ethanol, dioxane andtetrahydrofuran are preferable, and acetonitrile, ethyl acetate andethanol are particularly preferable.

Then, compound (IX) and a compound represented by the formula (X)[hereinafter referred to simply as compound (X)] are reacted to givecompound (Ia).

As the compound (IX), a crystal of an acid addition salt is preferablyused. In this reaction, generally 1-5 mol, preferably 1-2 mol, ofcompound (X) is reacted with 1 mol of compound (IX) in the presence ofan acid scavenger to capture the acid generated during the reaction, ina solvent that does not inhibit the reaction. As the solvent, forexample, tetrahydrofuran, acetonitrile, dioxane, acetone and a mixtureof these solvents with water are preferable, and acetonitrile,tetrahydrofuran, a mixture of acetonitrile with water, and a mixture oftetrahydrofuran with water are particularly preferable. Examples of theacid scavenger include ones generally used such as salts (e.g., sodiumhydrogencarbonate, sodium carbonate, potassium carbonate, sodiumacetate, potassium acetate, sodium phosphate and the like), tertiaryamines (e.g., triethylamine, tripropylamine, tributylamine,ethyldiisopropylamine, pyridine, lutidine, N,N-dimethylaniline,N-methylpiperidine, N-methylpyrrolidine, N-methylmorpholine and thelike), alkylene oxides (e.g., propyleneoxide, epichlorohydrin and thelike) and the like. Two or three therefrom may be used in a mixture. Ofthese, a combination of sodium hydrogencarbonate, sodium carbonate,sodium acetate, triethylamine or sodium acetate with triethylamine ispreferable, and particularly a combination of sodium hydrogencarbonate,sodium acetate, triethylamine or sodium acetate with triethylamine ispreferable. In this reaction, a dichlorophosphoryl group is successivelyhydrolyzed into phosphono group. The reaction temperature is from −40°C. to 80° C., preferably from −20° C. to 50° C. The reaction time is 20min-48 hr, preferably 30 min-24 hr. The compound (X) can be synthesizedby the method described in JP-A-11-255772 from the correspondingcompound represented by the formula (XI) [hereinafter referred to simplyas compound (XI)] and phosphorus pentachloride.

In this reaction, 1-5 mol, preferably 1 to 3 mol, of phosphoruspentachloride is generally reacted with 1 mol of compound (XI) in asolvent. As the solvent, tetrahydrofuran, ethyl acetate, isopropylether, dioxane, toluene and a mixture of these are preferable, andtetrahydrofuran, ethyl acetate, isopropyl ether and a mixture of theseare particularly preferable. The reaction temperature is from −40° C. to60° C., preferably from −10° C. to 25° C. The reaction time is 10 min-8hr, preferably 20 min-4 hr. For isolation after reaction, a generalmethod comprising extraction with water, or a method comprising directlyadding a poor solvent or a poor solvent and water to the reactionmixture to obtain precipitated solid can be utilized. The poor solventto be added is preferably toluene, isopropyl ether, n-hexane,cyclohexane or a mixture of these, particularly preferably toluene,isopropyl ether, n-hexane or a mixture of these.

EXAMPLES

The present invention is explained in detail by referring to thefollowing Reference Examples, Examples and Experimental Examples, whichare mere examples and do not limit the present invention. The presentinvention may be modified within the range that does not deviate fromthe scope of the present invention.

In the following Reference Examples, Examples and Experimental Examples,room temperature means 10-25° C.

The melting point was measured using YANAKO MP-J3. The ¹H-NMR spectrumwas measured using tetramethylsilane (CDCl₃, DMSO-d₆) or sodium3-trimethylsilyl-propionate-2,2,3,3-d₄(D₂O ) as an internal standard,and using VARIAN Gemini-200 (200 MHz), and all δ values are shown inppm.

For ultrasonic treatment, SHARP UT-204 (water bath type) and TAITECVP-60 (horn type) were used.

As the silica gel for column, kieselgel 60 (70-230 mesh, manufactured byMerck & Co., Inc.) was used. The column packing material ODS for HPLCwas manufactured by YMC Co., Ltd., and Diaion HP-20SS and SP-207 weremanufactured by Mitsubishi Chemical Corporation.

Elution in the column chromatography was carried out while monitoringwith TLC (Thin Layer Chromatography) or HPLC. In the TLC monitoring,60F254 manufactured by Merck & Co., Inc. was used as the TLC plate, asthe developing solvent, a solvent wherein the objective compound isdeveloped in the range of Rf value of 0.1-0.8 or a solvent similarthereto, and as the detection method, UV detection method was employed.For mixed solvents, the numeral value in the parenthesis is a mixingratio in volume of each solvent. The % for a solution indicates thenumber of g (grams) in 100 mL of the solution. In Reference Examples andExamples, the symbols mean the following.

s: singlet d: doublet t: triplet q: quartet ABq: AB type quartet dd:double doublet m: multiplet J: coupling constant

Example 1 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(100 mg, 0.151 mmol) was suspended in a mixture of distilled water forinjection (0.5 mL) and acetic acid (0.5 mL), and dissolved byultrasonication. This solution was stood overnight at room temperature.The precipitated crystals were pulverized with a spatula and collectedby filtration. The crystals were washed with distilled water forinjection (1.2 mL). Using molecular sieves 3A (1/16) as a drying agent,the crystals were dried under reduced pressure until they reached aconstant weight to give a seed crystal. yield: 79 mg (73%) meltingpoint: 221-223° C. (decomposition)

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(350 mg, 0.51 mmol) was suspended in a mixture of distilled water forinjection (2.38 mL) and acetic acid (2.38 mL), and dissolved byultrasonication. The seed crystal was added and crystallization wasallowed to take place with stirring at room temperature for 4 hr, whichwas followed by standing in a refrigerator overnight. A mixture (0.8 mL)of distilled water for injection/acetic acid (1:1) was added and theprecipitated crystals were pulverized with a spatula, collected byfiltration, washed 3 times with a mixture (0.8 mL) of distilled waterfor injection/acetic acid (1:1) and washed 5 times with distilled waterfor injection (1.0 mL). Using molecular sieves 3A (1/16) as a dryingagent, the crystals were dried under reduced pressure until they reacheda constant weight. yield: 260 mg (68%)

melting point: 221-223° C. (decomposition).

Anal Calcd for C₂₄H₂₅N₈O₁₀S₄P.1.0H₂O: C, 37.79, H, 3.57, N, 14.69, P,4.06. Found: C, 37.97, H, 3.30, N, 14.37, P, 3.88.

¹H-NMR (DMSO-d₆) δ: 1.24 (3H, t, J=7 Hz), 1.91 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.34 (3H, s), 5.32 (1H, d, J=5 Hz),5.92 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H,m), 9.70 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3202, 1755, 1668, 1645, 1537, 1392, 1273, 1039.

In FIG. 1, a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) of thecompound obtained in this Example is shown, wherein the transverse axisshows diffraction angle (2θ) and the vertical axis shows peak intensity.

Example 2 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(60 g, 87.6 mmol) was suspended in a mixture of distilled water forinjection (408 mL) and acetic acid (408 mL), and dissolved byultrasonication. The seed crystal obtained in Example 1 was added, andcrystallization was allowed to take place with stirring at roomtemperature for 5 hr, which was followed by standing in a refrigeratorovernight. The precipitated crystals were collected by filtration,washed twice with a mixture (68 mL) of distilled water forinjection/acetic acid (1:1) and 5 times with distilled water forinjection (85 mL). Using molecular sieves 3A (1/16) as a drying agent,the crystals were dried under reduced pressure until they reached aconstant weight. yield: 44.5g (68%)

melting point: 221-223° C. (decomposition).

Anal Calcd for C₂₄H₂₅N₈O₁₀S₄P.1.0H₂O: C 37.79, H 3.57, N 14.69, P 4.06.Found: C, 37.97, H, 3.30, N, 14.37, P, 3.88.

¹H-NMR (DMSO-d₆) δ: 1.24 (3H, t, J=7 Hz), 1.91 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.34 (3H, s), 5.32 (1H, d, J=5 Hz),5.92 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H,m), 9.70 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3202, 1755, 1668, 1645, 1537, 1392, 1273, 1039.

Example 3 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(58 g, 84.7 mmol) was suspended in a mixture of distilled water forinjection (208 mL) and acetic acid (208 mL), and dissolved byultrasonication. The seed crystal obtained in Example 1 was added andthe mixture was subjected to ultrasonication (horn type ultrasonicdevice) at room temperature for 30 min. A mixture (108 mL) of distilledwater for injection/acetic acid (1:1) was added, the crystals werepulverized and the mixture was stirred at room temperature for 1 hr. Amixture (108 mL) of distilled water for injection/acetic acid (1:1) wasadded, the crystals were pulverized and the mixture was stirred at roomtemperature for 1 hr. The above-mentioned step was repeated, which wasfollowed by standing in a refrigerator overnight. The precipitatedcrystals were collected by filtration, washed twice with a mixture (120mL) of distilled water for injection/acetic acid (1:1), and 5 times withdistilled water for injection (120 mL). Using diphosphorus pentaoxide asa drying agent, the crystals were dried under reduced pressure untilthey reached a constant weight. yield: 42.6g (68%)

melting point: 221-223° C. (decomposition).

Anal Calcd for C₂₄H₂₅N₈O₁₀S₄P.1.0H₂O: C, 37.79, H, 3.57, N, 14.69, P,4.06. Found: C, 37.97, H, 3.30, N, 14.37, P, 3.88.

¹H-NMR (DMSO-d₆) δ: 1.24 (3H, t, J=7 Hz), 1.91 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.34 (3H, s), 5.32 (1H, d, J=5 Hz),5.92 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H,m), 9.70 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3202, 1755, 1668, 1645, 1537, 1392, 1273, 1039.

Example 4 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(2.0 g, 2.92 mmol) was suspended in a mixture of distilled water forinjection (7.5 mL) and acetic acid (7.5 mL), and dissolved byultrasonication. The solution was stood at room temperature for 16 hr toallow crystallization to take place. The crystals were collected byfiltration, and washed twice with a mixture (5 mL) of distilled waterfor injection/acetic acid (1:1), and 5 times with distilled water forinjection (5 mL). Using molecular sieves 3A (1/16) as a drying agent,the crystals were dried under reduced pressure until they reached aconstant weight. yield: 1.41 g (65%)

melting point: 221-223° C. (decomposition).

Anal Calcd for C₂₄H₂₅N₈O₁₀S₄P.1.0H₂O: C, 37.79, H, 3.57, N, 14.69, P,4.06. Found: C, 37.97, H, 3.30, N, 14.37, P, 3.88.

¹H-NMR (DMSO-d₆) δ: 1.24 (3H, t, J=7 Hz), 1.91 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.34 (3H, s), 5.32 (1H, d, J=5 Hz),5.92 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H,m), 9.70 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3202, 1755, 1668, 1645, 1537, 1392, 1273, 1039.

Example 5 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith propionic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(100 mg, 0.15 mmol) was suspended in a mixture of distilled water forinjection (0.5 mL) and propionic acid (0.5 mL), and dissolved byultrasonication. Crystallization was allowed to take place with stirringat room temperature for 2.5 hr. The precipitated crystals were collectedby filtration, and washed with distilled water for injection (0.5 mL).Using diphosphorus pentaoxide as a drying agent, the crystals were driedunder reduced pressure until they reached a constant weight. yield: 97mg (88%)

melting point: 227-230° C. (decomposition).

Anal Calcd for C₂₅H₂₇N₈O₁₀S₄P.1.0H₂O: C, 38.66, H, 3.76, N, 14.16.Found: C, 38.51, H, 3.76, N, 14.16.

¹H-NMR (DMSO-d₆) δ: 0.99 (3H, t, J=7.6 Hz), 1.24 (3H, t, J=7 Hz), 2.21(2H, q, J=7.6 Hz), 3.59, 3.95 (2H, ABq, J=18 Hz), 4.17 (2H, q, J=7 Hz),4.33 (3H, s), 5.31 (1H, d, J=5 Hz), 5.91 (1H, dd, J=5&8 Hz), 8.52, 8.98(each 2H, d, J=6 Hz), 9.00 (1H, s), 9.24 (1H, m), 9.68 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3088, 1757, 1668, 1537, 1392, 1234, 1190, 1043.

In FIG. 2, a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) of thecompound obtained in this Example is shown, wherein the transverse axisshows diffraction angle (2θ) and the vertical axis shows peak intensity.

Example 6 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetonitrile

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(100 mg, 0.15 mmol) was suspended in a mixture of distilled water forinjection (1.2 mL) and acetonitrile (1.2 mL), and was dissolved byultrasonication and warming to 50° C. This solution was stood at roomtemperature overnight. The precipitated crystals were collected byfiltration, washed with water/acetonitrile (4:1), and air-dried to aconstant weight. yield: 63 mg (59%)

-   -   melting point: 210-215° C. (decomposition)

¹H-NMR (DMSO-d₆) δ: 1.23 (3H, t, J=7 Hz), 2.07 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.33 (3H, s), 5.32 (1H, d, J=5 Hz),5.91 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.34 (1H,m), 9.71 (1H, d, J=8 Hz).

In FIG. 3, a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) of thecompound obtained in this Example is shown, wherein the transverse axisshows diffraction angle (2θ) and the vertical axis shows peak intensity.

Example 7 Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

7β-Amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate(400 mg, 0.834 mmol) was dissolved in distilled water (24 mL) withstirring. 2M Aqueous sodium acetate solution (4.18 mL) was added withstirring under ice-cooling.2-(5-Dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylchloride (294 mg, 0.836 mmol) was dissolved in acetonitrile (2 mL) andadded at once to the above-mentioned reaction mixture with stirringunder ice-cooling. The mixture was stirred at room temperature for 1 hr15 min. Ethyl acetate (20 mL) was added to the reaction mixture forpartitioning. The separated aqueous layer was passed through a membranefilter (0.45 μm). The filtrate was concentrated to near dryness underreduced pressure. The residual solid was dissolved in distilled water (4mL). Ethanol (4 mL) was added and the mixture was stirred. The crystalsgradually precipitated. Ethanol (4 mL) was gradually added to promotecrystal growth. After the mixture was stood under ice-cooling for 30min, the precipitated crystals were collected by filtration. Thecrystals were washed successively with distilled water/ethanol (1:2, 4mL) and ethanol (4 mL), and air-dried on a funnel. yield: 554 mg (91%)

melting point: 217-222° C. (decomposition).

¹H-NMR (DMSO-d₆:D₂O=8:2) δ: 1.27 (3H, t, J=7 Hz), 3.40, 3.89 (2H, ABq,J=17 Hz), 4.24 (2H, q, J=7 Hz), 4.30 (3H, s), 5.23 (1H, d, J=5 Hz), 5.79(1H, d, J=5 Hz), 8.37, 8.77 (each 2H, d, J=7 Hz), 8.66 (1H, s).

IR (KBr) cm⁻¹: 3184, 1761, 1643, 1614, 1537, 1390, 1346, 1190, 1041.

In FIG. 4, a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) of thecompound obtained in this Example is shown, wherein the transverse axisshows diffraction angle (2θ) and the vertical axis shows peak intensity.

Example 8 Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

7β-Amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate(60 g, 125 mmol) was dissolved in distilled water (3.6 L) with stirring.2M Aqueous sodium acetate solution (700 mL) was added with stirringunder ice-cooling.2-(5-Dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylchloride (52.8 g, 150 mmol) was dissolved in acetonitrile (300 mL) andadded once to the above-mentioned reaction mixture with stirring underice-cooling. The mixture was stirred at room temperature for 1 hr. Ethylacetate (3 L) was added to the reaction mixture for partitioning. Theaqueous layer was separately taken and passed through a membrane filter(0.45 μm). The filtrate was concentrated under reduced pressure to 600mL. Ethanol (600 mL) was added and the crystals gradually precipitated.Ethanol (600 mL) was gradually added and the mixture was stood underice-cooling for 30 min. The precipitated crystals were collected byfiltration and washed successively with distilled water/ethanol (1:2,450 mL) and ethanol (600 mL). After washing, they were air-dried on afunnel. yield: 128.7 g (quantitative)

melting point: 217-222° C. (decomposition).

¹H-NMR (DMSO-d₆:D₂O=8:2) δ: 1.27 (3H, t, J=7 Hz), 3.40, 3.89 (2H, ABq,J=17 Hz), 4.24 (2H, q, J=7 Hz), 4.30 (3H,s), 5.23 (1H, d, J=5 Hz), 5.79(1H, d, J=5 Hz), 8.37, 8.77 (each 2H, d, J=7 Hz), 8.66 (1H, s).

IR (KBr) cm⁻¹: 3184, 1761, 1643, 1614, 1537, 1390, 1346, 1190, 1041.

Example 9 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(4.0 g, 5.84 mmol) was suspended in distilled water for injection (12mL), 2M aqueous sodium acetate solution (5.84 mL) was added, and themixture was dissolved. Acetic acid (24 mL) and then 1M sulfuric acid(5.78 mL) were added and a seed crystal was added. The mixture was stoodat room temperature for 24 hr. The precipitated crystals werepulverized, and collected by filtration. The obtained crystals werewashed 3 times with a mixture (4 mL) of distilled water forinjection/acetic acid (1:1), and 5 times with distilled water forinjection (4 mL). Using molecular sieves 3A (1/16) as a drying agent,the crystals were dried under reduced pressure until they reached aconstant weight. yield: 1.48 g (38%)

melting point: 221-223° C. (decomposition).

Anal Calcd for C₂₄H₂₅N₈O₁₀S₄P.1.0H₂O: C, 37.79, H, 3.57, N, 14.69, P,4.06. Found: C, 37.97, H, 3.30, N, 14.37, P, 3.88.

¹H-NMR (DMSO-d₆) δ: 1.24 (3H, t, J=7 Hz), 1.91 (3H, s), 3.58, 3.95 (2H,ABq, J=17 Hz), 4.17 (2H, q, J=7 Hz), 4.34 (3H, s), 5.32 (1H, d, J=5 Hz),5.92 (1H, dd, J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H,m), 9.70 (1H, d, J=8 Hz).

IR (KBr) cm⁻¹: 3202, 1755, 1668, 1645, 1537, 1392, 1273, 1039.

Example 10 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 9 except that distilled water forinjection was changed to 5% glucose injection, the title compound wasobtained. yield: 1.34 g (35%)

Example 11 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 9 except that distilled water forinjection was changed to 20% D-mannitol injection, the title compoundwas obtained. yield: 1.88 g (48%)

Example 12 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,12,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith Acetic Acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(4.0 g, 5.84 mmol) was suspended in 5% glucose injection (12 mL), and 2Maqueous sodium acetate solution (5.84 mL) was added for dissolution.Acetic acid (24 mL) and then 1M sulfuric acid (5.78 mL) were added and aseed crystal was added. The mixture was stirred at room temperature for24 hr. The precipitated crystals were collected by filtration, and theobtained crystals were washed 3 times with a mixture (4 mL) of distilledwater for injection/acetic acid (1:1) and 5 times with distilled waterfor injection (4 mL). Using molecular sieves 3A (1/16) as a dryingagent, the crystals were dried under reduced pressure until they reacheda constant weight. yield: 2.01 g (52%)

The physicochemical data were the same as those in Example 1.

Example 13 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 12 except that 2M aqueous sodiumacetate was changed to 2M aqueous ammonium acetate, the title compoundwas obtained. yield: 1.44 g (37%)

Example 14 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 12 except that distilled water forinjection was changed to 20% D-mannitol injection, the title compoundwas obtained. yield: 2.33 g (60%)

Example 15 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(10 g, 14.6 mmol) was suspended in 5% glucose injection (30 mL) and 2Maqueous sodium acetate solution (14.6 mL) was added for dissolution.Acetic acid (60 mL) and then 1M sulfuric acid (14.46 mL) were added anda seed crystal was added. The mixture was stirred at room temperaturefor 2 hr and stood for 22 hr. The precipitated crystals were pulverizedand collected by filtration. The obtained crystals were washed 3 timeswith a mixture (10 mL) of distilled water for injection/acetic acid(1:1) and 5 times with distilled water for injection (10 mL). Usingmolecular sieves 3A (1/16) as a drying agent, the crystals were driedunder reduced pressure until they reached a constant weight. yield: 6.90g (71%)

The physicochemical data were the same as those in Example 1.

Example 16 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(10 g, 14.6 mmol) was suspended in distilled water for injection (30 mL)and 2M aqueous sodium acetate solution (14.6 mL) was added fordissolution. Acetic acid (60 mL) and then 1M sulfuric acid (14.46 mL)were added and a seed crystal was added. The mixture was stirred at roomtemperature for 5 hr and stood for 1 hr. The precipitated crystals werecollected by filtration. The obtained crystals were washed 3 times witha mixture (10 mL) of distilled water for injection/acetic acid (1:1) and5 times with distilled water for injection (10 mL). Using molecularsieves 3A (1/16) as a drying agent, the crystals were dried underreduced pressure until they reached a constant weight. yield: 5.55 g(57%)

The physicochemical data were the same as those in Example 1.

Example 17 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 16 except that distilled water forinjection was changed to 5% glucose injection, the title compound wasobtained. yield: 5.70 g (59%)

Example 18 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

In the same manner as in Example 16 except that distilled water forinjection was changed to 20% D-mannitol injection, the title compoundwas obtained. yield: 6.21 g (64%)

Example 19 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(80 g, 116.8 mmol) was gradually added to a mixture of 20% D-mannitolinjection (160 mL) and 2M aqueous sodium acetate solution (116.8 mL) fordissolution. Acetic acid (400 mL) and then 1M sulfuric acid (115.7 mL)were added, and a seed crystal was added. The mixture was stirred atroom temperature for 3 hr. The precipitated crystals were collected byfiltration. The obtained crystals were washed 3 times with a mixture (80mL) of distilled water for injection/acetic acid (1:1) and 5 times withdistilled water for injection (160 mL). Using molecular sieves 3A (1/16)as a drying agent, the crystals were dried under reduced pressure untilthey reached a constant weight. yield: 50 g (64%)

The physicochemical data were the same as those in Example 1.

Example 20 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(1600 g, 2.34 mol) was dissolved in a solution of sodium acetate (428.2g, 5.14 mol), D-mannitol (425.7 g, 2.34 mol) and distilled water forinjection (6.1 L), and acetic acid (8 L) and 2M sulfuric acid (1864 mL,3.73 mol) were added. The mixture was stirred at room temperature for 30min, and a seed crystal (16 g) was added. The mixture was furtherstirred for 2 hr. The obtained crystals were collected by filtration,and washed with a mixture (20 L) of distilled water for injection/aceticacid (1:1). The crystals were through-flow dried until they reached aconstant weight. yield: 1390 g (74%)

Example 21 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(94.8 g, 138 mmol) was dissolved in 25% aqueous ammonia (10.4 g, 153mmol) and distilled water for injection (406 mL), and acetic acid (500mL) and 10% sulfuric acid (88.3 g, 90 mmol) were added. The seed crystal(80 mg) was added and the mixture was stirred at room temperature for2.5 hr. The mixture was stirred once every 30 min thereafter for thetotal of 5 hr, and stood overnight. A mixture (500 mL) of distilledwater for injection/acetic acid (1:1) was added. The crystals werecollected by filtration, and washed 3 times with a mixture (200 mL) ofdistilled water for injection/acetic acid (1:1). The crystals werethrough-flow dried until they reached a constant weight. yield: 75.5 g(71.5%)

The physicochemical data were the same as those in Example 1.

Example 22 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(10 g, 14.6 mol) was dissolved in a solution of 25% aqueous ammonia(2.18 g, 32.1 mmol), D-mannitol (2.66 g, 14.6 mol) and distilled waterfor injection (38 mL), and acetic acid (50 mL) and 2M sulfuric acid (12mL, 24.0 mol) were added. The mixture was stirred at room temperaturefor 30 min, and a seed crystal (0.1 g) was added. The mixture wasfurther stirred for 1.5 hr. The obtained crystals were collected byfiltration, and washed twice with a mixture (50 mL) of distilled waterfor injection/acetic acid (1:1), twice with a mixture (50 mL) ofdistilled water for injection/acetic acid (1:4), and once with a mixture(50 mL) of ethanol/acetic acid (1:1). The crystals were through-flowdried until they reached a constant weight. yield: 6.53 g (60%)

The physicochemical data were the same as those in Example 1.

Example 23 Crystal of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid

An aqueous solution of disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylateobtained by column chromatography according to the method of ReferenceExample 25 was concentrated under reduced pressure to give 52.2 gthereof (content 19.2%, 13.7 mmol). Acetic acid (52.2 mL) and 1Msulfuric acid (27.4 mL, 27.4 mmol) were added to the solution. A seedcrystal was added, and the mixture was stirred at room temperature for 5hr and stood for 1 hr. The crystals were collected by filtration, andwashed with a mixture (100 mL) of distilled water for injection/aceticacid (1:1) and distilled water for injection (200 mL). The crystals weredried in vacuo until they reached a constant weight. yield: 7.02 g(68.8%)

Example 24

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticAcid Solvate

To a solution of sodium acetate (1001 g, 12.2 mol) in distilled waterfor injection (15 L) was added3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(3740 g, 5.46 mol) for dissolution. The mixture was passed through a 0.2μm membrane filter, and washed with distilled water for injection (9 L).To a mixture of the filtrate and washing were added acetic acid (28 L)and then 2M sulfuric acid (4.35 L) and a seed crystal (3.14 g) wasadded. The mixture was stirred at 30° C. for 5 hr. The precipitatedcrystals were collected by filtration, and washed with a mixture (75 L)of distilled water for injection/acetic acid (1:1), a mixture (19 L) ofdistilled water for injection/acetic acid (1:4) and a mixture (19 L) ofethanol/acetic acid (1:1) with stirring. The air having a dew point of−5° C. was passed to dry the crystals. yield: 2011 g (49%)

In FIG. 5, a powder X-ray diffraction spectrum (Cu, 40 kV, 50 mA) of thecompound obtained in this Example is shown, wherein the transverse axisshows diffraction angle (2θ) and the vertical axis shows peak intensity.

Reference Example 1

Benzhydryl7β-amino-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate

Benzhydryl7β-phenylacetylamino-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate(4.15 g, 6.0 mmol) was dissolved in dichloromethane (60 mL) and pyridine(0.726 mL, 9.0 mmol) and phosphorus pentachloride (1.87 g, 9.0 mmol)were successively added under ice-cooling. The mixture was stirred underice-cooling for 1 hr. Isobutanol (8.0 mL) was added at once to thereaction mixture, and the mixture was stirred at room temperature for 1hr. Isopropyl ether (300 mL) was added dropwise and the mixture wasstirred for 10 min. The solvent was removed by decantation. The residualoil was suspended in ethyl acetate (600 mL) and saturated aqueous sodiumhydrogencarbonate solution (200 mL) was added dropwise, after which themixture was stirred for 15 min. The organic layer was separately taken,dried over magnesium sulfate and filtrated. The solvent was evaporatedoff under reduced pressure. Isopropyl ether (60 mL) was added to theresidue, and the precipitated powder was collected by filtration, washedwith isopropyl ether (20 mL) and dried under reduced pressure. yield:3.18 g (95%)

¹H-NMR (DMSO-d₆) δ: 3.51, 3.77 (2H, ABq, J=18 Hz), 4.82 (1H, d, J=5 Hz),5.02 (1H, d, J=5 Hz), 7.01 (1H, s), 7.2-7.5 (10H, m), 7.71 (1H, s),7.73, 8.68 (each 2H, d, J=6 Hz).

Reference Example 2

Benzhydryl7β-t-butoxycarbonylamino-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate

Benzhydryl7β-amino-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate (3.0g, 5.37 mmol) was suspended in tetrahydrofuran (40 mL) and dibutyldicarbonate (2.34 g, 10.7 mmol) was added. The mixture was stirred atroom temperature for 18 hr and concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate (30 mL) and applied to silica gelcolumn (30 g). The fractions containing the title compound eluted withethyl acetate were collected, and the solvent was evaporated off underreduced pressure. Isopropyl ether (50 mL) was added to the residue, andthe precipitated powder was collected by filtration, washed withisopropyl ether (10 mL) and dried under reduced pressure. yield: 1.4 g(40%)

¹H-NMR (CDCl₃) δ: 1.45 (9H, s), 3.51, 3.74 (2H, ABq, J=18 Hz), 5.04 (1H,d, J=5 Hz), 5.40 (1H, d, J=10 Hz), 5.69 (1H, dd, J=5&10 Hz), 7.00 (1H,s), 7.1-7.5 (10H, m), 7.70-7.74 (3H, m), 8.67 (2H, d, J=6 Hz).

Reference Example 3

Benzhydryl7β-t-butoxycarbonylamino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateIodide

Benzhydryl7β-t-butoxycarbonylamino-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate(1.3 g, 1.97 mmol) was dissolved in dimethylformamide (2.6 mL) andiodomethane (1.23 mL, 19.7 mmol) was added. The mixture was stirred atroom temperature for 5 hr. The reaction mixture was concentrated underreduced pressure and diethyl ether (100 mL) was added to the residue.The mixture was stirred for 10 min. The precipitated powder wascollected by filtration, washed with diethyl ether (20 mL) and driedunder reduced pressure. yield: 1.57 g (99%)

¹H-NMR (DMSO-d₆) δ: 1.41 (9H, s), 3.68, 3.97 (2H, ABq, J=18 Hz), 4.34(3H, s), 5.29 (1H, d, J=5 Hz), 5.67 (1H, dd, J=5&8.6 Hz), 6.96 (1H, s),7.1-7.5 (10H, m), 8.16 (1H, d, J=8.6 Hz), 8.53, 9.00 (each 2H, d, J=6.6Hz), 9.02 (1H, s).

Reference Example 4

7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate.dihydrochloride

Benzhydryl7β-t-butoxycarbonylamino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateiodide (1.3 g, 1.62 mmol) was dissolved in acetonitrile (3 mL), andconc. hydrochloric acid (3 mL) was added. The mixture was stirred at35-40° C. for 2 hr. The reaction mixture was concentrated under reducedpressure, and ethanol (30 mL) was added to the residue. The precipitatedpowder was collected by filtration, washed with ethanol (10 mL) anddried under reduced pressure. yield: 239 mg (31%)

¹H-NMR (DMSO-d₆) δ: 3.82, 3.96 (2H, ABq, J=18 Hz), 4.35 (3H, s), 5.27(1H, d, J=5 Hz), 5.40 (1H, d, J=5 Hz), 8.61, 9.06 (each 2H, d, J=6 Hz),9.16 (1H, s).

Reference Example 5

4-(4-pyridyl)-1,3-thiazole-2-thiol Sodium Salt

4-(4-Pyridyl)-1,3-thiazole-2-thiol (194 g, 1.0 mol, powder) was added to8N aqueous sodium hydroxide solution (1.25 L, 10 mol) and the mixturewas stirred for 30 min. The precipitated crystals were collected byfiltration and washed with 8N aqueous sodium hydroxide solution (0.4 L).The obtained wet crystals were recrystallized from isopropyl alcohol(200 mL) to give the title compound (166 g, 0.77 mol) as yellowcrystals. yield 77%.

melting point: 272° C. (decomposition).

Anal Calcd for C₈H₅N₂S₂Na.0.75H₂O: C 41.82, H 2.85, N 12.19. Found: C41.78, H 2.98, N 12.11.

¹H-NMR(DMSO-d₆)δ:7.35 (1H, s), 7.71 (2H, d, J=6.2 Hz), 8.48 (2H, d,J=6.2 Hz).

Reference Example 6

4-(4-pyridyl)-1,3-thiazole-2-thiol Sodium Salt

4-(4-Pyridyl)-1,3-thiazole-2-thiol (194 g, 1.0 mol) was suspended inmethanol (1 L) and a powder of sodium methylate (71.5 g, 1.2 mol) wasadded at 25° C. The mixture was stirred for 30 min and the reactionmixture was concentrated under reduced pressure to 50-60 mL. The mixturewas preserved overnight in a refrigerator. The precipitated crystalswere collected by filtration and dried in vacuo in the presence ofphosphorus pentoxide at 400C to give the title compound (160 g, 0.74mol). yield: 74%.

Reference Example 7

Benzhydryl7β-[(phenylacetyl)amino]-3-[(methylsulfonyl)oxy]-3-cephem-4-carboxylate

Benzhydryl 7β-[(phenylacetyl)amino]-3-hydroxy-3-cephem-4-carboxylate(500 g, 1 mol) was dissolved in acetonitrile (2 L) andethyldiisopropylamine (183 mL, 1.05 mol) was added dropwise over 10 minwith stirring at −40° C. Then, methanesulfonyl chloride (86 mL, 1.1 mol)was added dropwise over 10 min and the mixture was stirred at −40° C.for 40 min. The reaction mixture was poured into iced water (8 L) andthe precipitate was collected by filtration. The precipitate was washedwith water (2 L) and ethyl acetate (300 mL) and dried under reducedpressure to give the title compound (544 g, 0.94 mol) as pale-yellowcrystals. yield: 94%.

melting point: 157° C.

Anal Calcd for C₂₉H₂₆N₂O₇S₂: C 60.19, H 4.53, N 4.84, S 11.08. Found: C59.86, H 4.72, N 4.73, S 10.77.

¹H-NMR(CDCl₃)δ:2.79 (3H, s), 3.48-3.75 (4H, m), 5.02 (1H, d, J=5.2 Hz),5.90 (1H, dd, J=5.2 Hz, 8.8 Hz), 6.24 (1H, d, J=8.8 Hz), 6.93 (1H, s),7.24-7.41 (15H, m)

Reference Example 8

Benzhydryl7β-[(phenylacetyl)amino]-3-[(methylsulfonyl)oxy]-3-cephem-4-carboxylate

Benzhydryl 7β-[(phenylacetyl)amino]-3-hydroxy-3-cephem-4-carboxylate(500 g, 1 mol) was dissolved in acetone (2 L) and methanesulfonylchloride (86 mL, 1.1 mol) was added dropwise over 10 min with stirringat −20° C. Then, ethyldiisopropylamine (183 mL, 1.05 mol) was addeddropwise over 30 min and the mixture was stirred at −20° C. for 40 min.The reaction mixture was poured into iced water (8 L) and theprecipitate was collected by filtration. The precipitate was then washedwith water (2 L) and ethyl acetate (300 mL), and dried under reducedpressure to give the title compound (523 g, 0.90 mol). yield: 90%.

Reference Example 9

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-pyridyl-2-thiazolylthio]-3-cephem-4-carboxylate

4-(4-Pyridyl)-1,3-thiazole-2-thiol sodium salt (194 g, 1 mol) preparedaccording to the method of Reference Example 5 was suspended intetrahydrofuran (1.5 L) and benzhydryl7β-[(phenylacetyl)amino]-3-[(methylsulfonyl)oxy]-3-cephem-4-carboxylate(196 g, 0.91 mol) dissolved in tetrahydrofuran (3.0 L) was addeddropwise under ice-cooling over 30 min. The mixture was stirred at 0° C.for 2 hr and saturated brine (7 L) was added. The mixture was extractedwith ethyl acetate (5 L). The organic layer was washed with saturatedbrine (5 L) and dried over anhydrous sodium sulfate. The solvent wasconcentrated under reduced pressure to give the title compound ascrystals. The crystals were suspended in ethyl acetate (0.5 L),collected by filtration on a glass filter and washed with methanol (1L×2). The crystals were dried in vacuo to give the title compound (412g, 0.71 mol). yield: 78%.

melting point: 134° C.

Anal Calcd for C₃₆H₂₈N₄O₄S₃.0.5H₂O: C 63.05, H 4.11, N 8.17, S 14.02.Found: C 63.16, H 4.15, N 8.27, S 13.98.

¹H-NMR(CDCl₃)δ:3.41-3.73 (4H, m), 5.02 (1H, d, J=4.8 Hz), 5.84 (1H, dd,J=4.8 Hz, 8.8 Hz), 6.23 (1H, d, J=8.8 Hz), 6.97 (1H, s), 7.27-7.72 (17H,m), 7.73 (1H, s), 8.67 (1H, d, J=6.2 Hz).

Reference Example 10

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-pyridyl-2-thiazolylthio]-3-cephem-4-carboxylate

4-(4-Pyridyl)-1,3-thiazole-2-thiol (225 g, 1.16 mol) was suspended intetrahydrofuran (1.5 L) and 28% sodium methylatemethanol solution (235g, 1.22 mol) was added dropwise at 25° C. over 10 min. The mixture wasstirred for 1 hr. The reaction mixture was ice-cooled and benzhydryl7β-[(phenylacetyl)amino]-3-[(methylsulfonyl)oxy]-3-cephem-4-carboxylate(479 g, 0.83 mol) dissolved in tetrahydrofuran (3.5 L) was addeddropwise over 30 min. The mixture was stirred at 0° C. for 1 hr and amixture of acetic acid (50 mL), methanol (5 L) and water (7 L) was addeddropwise over 30 min. The mixture was stirred at 0° C. for 2 hr, and theprecipitated crystals were collected by filtration. The obtainedcrystals were washed with methanol (1 L×2) and dried in vacuo to givethe title compound (438 g, 0.63 mol). yield: 76%.

Reference Example 11

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateIodide

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-pyridyl-2-thiazolylthio]-3-cephem-4-carboxylate(300 g, 0.43 mol) was dissolved in tetrahydrofuran (0.6 L) and methyliodide (324 g, 2.17 mol) was added at 25° C. After stirring for 8 hr,the reaction mixture was poured into ethyl acetate (6 L). Theprecipitate was collected by filtration. The obtained precipitate waswashed with ethyl acetate (0.5 L) and diethyl ether (1 L), and dried invacuo to give the title compound (351 g, 0.42 mol) as a yellow powder.yield: 97%.

¹H-NMR(CDCl₃)δ:3.58 (2H,dd,J=4.8Hz,6.6Hz), 3.74 (2H,brs), 4.35 (3H, s),5.11 (1H, d, J=4.8 Hz), 5.89 (1H, d, J=4.8 Hz), 6.95 (1H, s), 7.18-7.42(15H, m), 8.30 (2H, d, J=6.6 Hz), 8.41 (1H, s), 8.75 (2H, d, J=6.6 Hz).

Reference Example 12

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateIodide

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-pyridyl-2-thiazolylthio]-3-cephem-4-carboxylate(300 g, 0.43 mol) was dissolved in N,N-dimethylformamide (0.6 L), andmethyl iodide (648 g, 4.34 mol) was added at 250C. After stirring for 16hr, the reaction mixture was poured into ethyl acetate (6 L), and theprecipitate was collected by filtration. The precipitate was washed withethyl acetate (0.5 L) and diethyl ether (1 L), and dried in vacuo togive the title compound (317 g, 0.39 mol) as a yellow powder. yield:89%.

Reference Example 13

Benzhydryl7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateChloride Monohydrochloride

Phosphorus pentachloride (312 g, 1.44 mol) was suspended indichloromethane (2.8 L), and pyridine (115 g, 1.44 mol) was addeddropwise under ice-cooling over 10 min. The mixture was stirred for 30min. Then, a powder (400 g, 0.48 mol) of benzhydryl7β-[(phenylacetyl)amino]-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateiodide was added over 10 min and the mixture was stirred for 1 hr. Thereaction mixture was cooled to −10° C. and isobutyl alcohol (5.6 L) wasadded. The mixture was stirred at 25° C. for 3 hr. Then ethyl acetate (6L) was added and the mixture was further stirred for 3 hr. Theprecipitate was collected by filtration on a glass filter, washed withethyl acetate (0.5 L) and diethyl ether (1 L), and dried in vacuo togive the title compound (270 g, 0.42 mol) as a pale-yellow powder.yield: 87%.

¹H-NMR(DMSO-d₆)δ:3.94 (2H, brs), 4.35 (3H, s), 5.32 (1H, d, J=5.0 Hz),5.45 (1H, d, J=5.0 Hz), 6.99 (1H, s), 7.25-7.40 (10H, m), 8.59 (2H, d,J=7.0 Hz), 9.07 (2H, d, J=7.0 Hz), 9.19 (1H, s).

Reference Example 14

7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateDihydrochloride

Benzhydryl7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylatechloride monohydrochloride (430 g, 0.66 mol) was suspended inacetonitrile (3.5 L) and conc. hydrochloric acid (3.5 L) was added. Themixture was stirred for 10 min. Then, ethyl acetate (7 L) was added tothe reaction mixture, and the mixture was stirred for 5 hr. Theprecipitate was collected by filtration, washed with acetonitrile(1L×2), and dried in vacuo to give the title compound (236 g, 0.49 mol)as pale-yellow crystals. yield: 74%.

melting point: 202° C. (decomposition).

Anal Calcd for C₁₆H₁₄N₄O₃S₃.2HCl: C 40.08, H 3.36, N 11.69, S 20.07.Found: C 39.83, H 3.43, N 11.78, S 20.03.

¹H-NMR(DMSO-d₆)δ:3.90 (2H, dd, J=17.2 Hz, 17.6 Hz), 4.35 (3H, s), 5.26(1H, d, J=5.0 Hz), 5.42 (1H, d, J=5.0 Hz), 8.61 (2H, d, J=7.0 Hz), 9.05(2H, d, J=7.0 Hz), 9.17 (1H, s).

Reference Example 15

7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateDihydrochloride

Phosphorus pentachloride (31.2 g, 144 mmol) was suspended indichloromethane (250 mL) and pyridine (11.5 g, 144 mmol) was addeddropwise under ice-cooling over 10 min. The mixture was stirred for 30min. Then, a powder (41.7 g, 48 mmol) of benzhydryl7β-[(phenylacetyl)amino]-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateiodide was added over 10 min, and the mixture was stirred for 1 hr. Thereaction mixture was cooled to −10° C. and isobutyl alcohol (250 mL) wasadded. The mixture was stirred at 25° C. for 3 hr. The reaction mixturewas concentrated to about 100 mL and acetonitrile (250 mL) and thenconc. hydrochloric acid (250 mL) were added. The mixture was stirred at40° C. for 2 hr. The precipitate was collected by filtration, washedwith acetonitrile (100 mL×2), and dried in vacuo to give the titlecompound (14.5 g, 0.30 mol). yield: 63%.

Reference Example 16

7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylateMonohydrochloride

Lyophilized product (5.8 g, 14.3 mmol) of7β-amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylatewas dissolved in water (290 mL) and 1N hydrochloric acid was added underice-cooling to adjust pH to 1.3. The solution was concentrated to about30 mL under reduced pressure, and ethanol (70 mL) was gradually addedunder ice-cooling with shaking. The mixture was stood under ice-coolingfor 2 hr. The precipitated crystals were collected by filtration, washedwith ethanol/water (5:1, 30 mL) and dried under reduced pressure. yield:4.1g (65%)

melting point: 120-140° C. (decomposition).

Anal Calcd for C₁₆H₁₅N₄O₃S₃Cl.3.0H₂O: C 38.67, H 4.26, N 11.27, Cl 8.00.Found: C 38.58, H 3.92, N 11.26, Cl 8.18.

¹H-NMR (D₂O ) δ: 3.64, 3.99 (2H, ABq, J=18 Hz), 4.37 (3H, s), 5.23 (1H,d, J=5 Hz), 5.44 (1H, d, J=5 Hz), 8.31, 8.76 (each 2H, d, J=7 Hz),8.51(1H, s).

IR (KBr) cm⁻¹: 3400, 1800, 1770, 1640, 1530, 1405, 1330, 1190, 1020.

Reference Example 17

2-(5-dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylChloride

Phosphorus pentachloride (1.46 g, 7.0 mmol) was suspended in ethylacetate (4.17 mL) and the mixture was stirred under ice-cooling for 5min. 2-(5-Amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetic acid (600mg, 2.77 mmol) was added at once with stirring under ice-cooling fordissolution. The mixture was stirred under ice-cooling for 30 min. Thereaction mixture was diluted with toluene (16.8 mL) under ice-coolingfor dissolution. To this solution was added saturated brine (11.1 mL)cooled to not higher than −5° C., and the mixture was stirred underice-cooling for 5 min. The reaction mixture was transferred to aseparating funnel and the organic layer was separately taken withoutshaking. The organic layer was dried over magnesium sulfate. Afterfiltration, the mother liquor was evaporated off under reduced pressureand the residue was stood under ice-cooling for 30 min (crystalsprecipitated from the residual oil). Diisopropyl ether/n-hexane (1:1,5.67 mL) was added and the crystals were pulverized with a spatula. Themixture was stood under ice-cooling for 15 min. The precipitatedcrystals were collected by filtration, washed with diisopropylether/n-hexane (1:1, 5.67 mL) and dried under reduced pressure. yield:631 mg (64%)

melting point: 116-119° C.

Anal Calcd for C₆H₆N₄O₃SCl₃P: C 20.50, H 1.72, N 15.94, P 8.81. Found: C20.52, H 1.77, N 15.99, P 8.90.

¹H-NMR (CDCl₃) δ: 1.42 (3H, t, J=7 Hz), 4.45 (2H, q, J=7 Hz), 8.81 (1H,br s).

IR (KBr) cm⁻¹: 3063, 2984, 1784, 1593, 1223, 1057.

Reference Example 18

2-(5-dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylChloride

Phosphorus pentachloride (78 g, 375 mmol) was suspended in ethyl acetate(225 mL) and 2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoaceticacid (32.4 g, 150 mmol) was added with stirring under ice-cooling. Themixture was stirred under ice-cooling for 30 min. The reaction mixturewas diluted with toluene (900 mL) under ice-cooling for dissolution. Tothis solution was added saturated brine (600 mL) cooled to not higherthan −5° C., and the mixture was stirred under ice-cooling for 10 min.The reaction mixture was transferred to a separating funnel and theorganic layer was separately taken. The organic layer was dried overmagnesium sulfate. After filtration, the mother liquor was evaporatedoff under reduced pressure (crystals precipitated). Diisopropyl ether(300 mL) was added and the crystals were pulverized with a spatula. Themixture was stirred under ice-cooling for 30 min and stood for 30 min.The precipitated crystals were collected by filtration, washed withdiisopropyl ether (20 mL) and dried under reduced pressure. yield: 23.5g (45%)

melting point: 116-119° C.

Anal Calcd for C₆H₆N₄O₃SCl₃P: C 20.50, H 1.72, N 15.94, P 8.81. Found: C20.52, H 1.77, N 15.99, P 8.90.

¹H-NMR (CDCl₃) δ: 1.42 (3H, t, J=7 Hz), 4.45 (2H, q, J=7 Hz), 8.81 (1H,br s).

IR (KBr) cm⁻¹: 3063, 2984, 1784, 1593, 1223, 1057.

Reference Example 19

2-(5-dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylChloride

2-(5-Amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetic acid (1.0 kg,4.6 mol) was suspended in ethyl acetate (2.4 L) and diisopropyl ether(1.6 L), and cooled to not higher than −1° C. with nitrogen gassubstitution. Phosphorus pentachloride (2.0 kg, 9.6 mol) was added atnot higher than 5° C. and the mixture was stirred for 30 min.Diisopropyl ether (2.0 L), water (120 mL) and n-hexane (12 L) were addedat not higher than 2° C. and the mixture was stirred at the sametemperature for 1 hr. The precipitated crystals were collected byfiltration under a nitrogen stream, washed with diisopropylether/n-hexane (1:2, 2.0 L) and n-hexane (2.0 L) and dried bythrough-flow under a nitrogen stream. yield: 876.0 g (49.7%)

Reference Example 20

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(1.43 g, 1.96 mmol) was gradually dissolved in 1%(v/v) aqueous aceticacid (14 mL) at room temperature. The solution was filled in an SP-207column (60 mL). After successive elution with 2% (w/v) brine (180 mL)and 3% (v/v) aqueous ethanol (60 mL), a fraction (1500 mL) containingthe title compound eluted with 10% (v/v) aqueous ethanol was collectedand concentrated to about 20 mL under reduced pressure. 6N Hydrochloricacid (about 1.5 mL) was gradually added with shaking under ice-coolingto adjust to pH 0.5. A white powder was precipitated. This was stoodunder ice-cooling for 30 min and the precipitated powder was collectedby filtration and washed 3 times with distilled water (2 mL). Usingmolecular sieves 3A (1/16) as a drying agent, the powder was dried underreduced pressure to a constant weight. yield: 800 mg (59%)

Anal Calcd for C₂₂H₂₁N₈O₈S₄P.2.0H₂O: C 36.66, H 3.50, N 15.55, P 4.30.Found: C 36.94, H 3.46, N 15.57, P 3.95.

¹H-NMR (DMSO-d₆) δ: 1.23 (3H, t, J=7 Hz), 3.58, 3.94 (2H, ABq, J=18 Hz),4.17 (2H, q, J=7 Hz), 4.33 (3H, s), 5.32 (1H, d, J=5 Hz), 5.90 (1H, dd,J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H, m), 9.70 (1H,d, J=8 Hz).

IR (KBr) cm⁻¹: 3055, 1778, 1682, 1643, 1520, 1385, 1190, 1038.

Reference Example 21

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate(128 g, 1.96 mmol) was gradually dissolved in 1% (v/v) aqueous aceticacid (1.28 L) at room-temperature. The solution was filled in an SP-207column (2.5 L). After elution with 1% (v/v) aqueous acetic acid (9 L), afraction (32 L) containing the title compound eluted with 0.1M sodiumacetate:0.1M acetic acid:ethanol (960:30:110) was collected andconcentrated to about 700 mL under reduced pressure. 6N Hydrochloricacid (480 mL) was gradually added with shaking under ice-cooling toadjust pH to 0.5. A white powder was precipitated. This was stood underice-cooling for 30 min and the precipitated powder was collected byfiltration and washed with distilled water (300 mL). Using molecularsieves 3A (1/16) as a drying agent, the powder was dried under reducedpressure to a constant weight. yield: 60.9 g (68%)

Anal Calcd for C₂₂H₂₁N₈O₈S₄P.2.0H2O: C 36.66, H 3.50, N 15.55, P 4.30.Found: C 36.94, H 3.46, N 15.57, P 3.95.

¹H-NMR (DMSO-d₆) δ: 1.23 (3H, t, J=7 Hz), 3.58, 3.94 (2H, ABq, J=18 Hz),4.17 (2H, q, J=7 Hz), 4.33 (3H, s), 5.32 (1H, d, J=5 Hz), 5.90 (1H, dd,J=5&8 Hz), 8.51 (2H, d, J=6 Hz), 8.99 (3H, m), 9.30 (1H, m), 9.70 (1H,d, J=8 Hz).

IR (KBr) cm⁻¹: 3055, 1778, 1682, 1643, 1520, 1385, 1190, 1038.

Reference Example 22

disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

7β-Amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate1.30 kg (2.71 mol) was suspended in water (7.80 L) and 3M sodium acetate(1.81 L, 5.42 mol) and triethylamine (2.0 L, 14.4 mol) were successivelyadded under ice-cooling.2-(5-Dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylchloride (1143.7 g, 3.25 mol) was dissolved in tetrahydrofuran (3.1 L)and the solution was cooled to not higher than −20° C. and added to theabove-mentioned reaction mixture. The temperature of the mixture wasraised to 15-25° C., and 3M sodium acetate (5.78 L, 17.4 mol) and ethylacetate (6.50 L) were successively added for partitioning. Ethanol (30L) was added dropwise to the aqueous layer, and after ice-cooling, theprecipitated powder was collected by filtration and washed successivelywith water/ethanol (1:2, 6.5 L) and ethanol (13 L). After through-flowdrying, the powder was dissolved in diluted brine (19.5 L) and thesolution was used as a stock solution for column chromatography. yield:23.3 kg (content 6.61%, yield: 78%)

Reference Example 23

disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

7β-Amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate(5.0 g, 10.41 mmol) was suspended in water/tetrahydrofuran (5:1, 30 mL)and 3M sodium acetate (6.9 mL, 20.8 mmol) was added dropwise.Triethylamine (7.2 mL, 52.0 mmol) was added under ice-cooling.2-(5-Dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylchloride (5.87 g, 16.7 mmol) was dissolved in tetrahydrofuran (12 mL)and added dropwise to the above-mentioned reaction mixture. Thetemperature of the mixture was raised to 15-30° C., and 3M sodiumacetate (28.4 mL, 85.3 mmol) and ethyl acetate (25 mL) were successivelyadded for partitioning. Ethanol (120 mL) was added dropwise to theaqueous layer, and after ice-cooling, the precipitated powder wascollected by filtration, washed successively with water/ethanol (1:2)and ethanol, and dried by through-flow. yield: 7.78 g (content 66.2%,yield: 70%)

Reference Example 24

Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

7β-Amino-3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-3-cephem-4-carboxylate(5.00 g, 10.4 mmol) was suspended in water/acetonitrile (10:1, 33 mL)and a part of triethylamine (19.1 mL, 70.7 mmol) was added underice-cooling.2-(5-Dichlorophosphorylamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetylchloride (5.87 g, 16.6 mmol) was dissolved in acetonitrile (12 mL) andadded dropwise to the above-mentioned reaction mixture while theremainder of triethylamine was added dropwise. The temperature of themixture was raised to 25° C., and 3M sodium acetate (19.1 mL, 57.2 mmol)and ethyl acetate (25 mL) were successively added for partitioning.Ethanol (120 mL) was added dropwise to the aqueous layer, and afterice-cooling, the precipitated powder was collected by filtration. Thepowder was washed successively with water/ethanol (1:2, 25 mL) andethanol (50 mL), and dried by through-flow. yield: 6.78 g (content73.1%, yield: 68%)

Reference Example 25

Purification of Disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

An aqueous solution (7.33 kg, content 4.98%, 0.516 mol) of disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewas applied to SP-207 column chromatography (18 L) and successivelyeluted with diluted brine and aqueous ethanol. The resulting mainlyeluted solution was concentrated by evaporator. recovery: 2.16 kg(content 14.4%, yield: 83%)

Reference Example 26

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

Activated carbon (15.3 g) was added to an aqueous solution (1.89 kg,content 16.1%, 0.419 mol) of disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylateand the mixture was stirred. After filtering off the activated carbon,the residue was washed with water. Water was added to the filtrate to3.96 kg. Acetic acid (95.0 mL, 1.68 mol) was added and ethanol (4 L) wasadded. 6N Hydrochloric acid (154 mL, 0.922 mol) was added and themixture was ice-cooled. The precipitated powder was collected byfiltration. The powder was successively washed with water/ethanol(1.0:1.1, 0.71 L) and ethanol (2.1 L) and dried by through-flow. yield:249.7 g (content 90.8%, yield: 80%)

Reference Example 27

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

Water was added to an aqueous solution (57.3 g, content 8.73%, 6.86mmol) of disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylateto 65 g. Acetic acid (1.57 mL, 26.2 mmol) and ethanol (65 mL) were addedat room temperature and 10% sulfuric acid (8.7 mL, 8.88 mmol) was addeddropwise. After stirring under ice-cooling, the precipitated powder wascollected by filtration. The powder was successively washed withwater/ethanol (1:1, 10 mL) and ethanol (30 mL) and dried in vacuo.yield: 3.8 g (81%)

Reference Example 28

3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate

Water was added to an aqueous solution (664 g, content 12.1%, 0.114 mol)of disodium3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonateamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylateto 1044 g. 6N Hydrochloric acid (56.9 mL, 0.341 mol) was added dropwiseat not higher than 10° C., and after stirring, the precipitated powderwas collected by filtration. The powder was washed with water (563 mL)and dried in vacuo. yield: 81.4 g (content 81.6%, yield 81%)

Reference Example 29

4-(4-pyridyl)-1,3-thiazole-2-thiol

4-(4-Pyridyl)-1,3-thiazole-2-thiol.hydrobromide (89.3 g, 0.32 mol) wassuspended in water (627 mL) under a nitrogen stream, and 25% aqueoussodium hydroxide solution (110.5 g, 0.69 mol) was added for dissolution.An insoluble material was filtered and washed with water (100 mL). 35%Hydrochloric acid (31 mL) was added to the filtrate to adjust to pH 6.8.The precipitated crystals were collected by filtration, and washed withwater (20 mL) and methanol (20 mL). The obtained crystals were suspendedin methanol (627 mL). After stirring for 2 hr, the crystals werefiltrated and dried. yield: 47.6 g (75.6%)

Reference Example 30

Benzhydryl7β-[(phenylacetyl)amino]-3-[4-(4-pyridyl)-2-thiazolylthio]-3-cephem-4-carboxylate

Benzhydryl7β-[(phenylacetyl)amino]-3-[(methylsulfonyl)oxy]-3-cephem-4-carboxylate-(900g, 1.56 mol) was dissolved in tetrahydrofuran (3.6 L) and cooled to −3°C. While maintaining the same temperature, 28% sodium methylate-methanolsolution (360 g, 1.87 mol) of 4-(4-pyridyl)-1,3-thiazole-2-thiol (362.5g, 1.87 mol) obtained in the same manner as in Reference Example 29, andtetrahydrofuran (720 mL) solution were added. The mixture was stirredfor 1.5 hr. Acetic acid (18.7 g) was added and after stirring for 30min, methanol (9 L) and water (5.4 L) were added. The mixture wasstirred for 2 hr. The precipitated crystals were collected byfiltration, washed with methanol (16 L) and dried in vacuo. yield: 884 g(84%)

Experimental Example 1

The crystals (102.4 mg, 0.131 mmol) of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith acetic acid, and sodium hydrogencarbonate (27.6 mg, 0.328 mmol)were filled in a vial, and then a physiological saline solution (0.918mL) was gradually added. These compounds dissolved while generatingcarbon dioxide gas and gave a clear solution. This solution was dilutedwith a physiological saline solution to 2.0 mL, whereby a medicationsolution having a concentration of 50 mg/mL was prepared.

Experimental Example 2

The crystals (101.9 mg, 0.129 mmol) of3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylatewith propionic acid, and sodium hydrogencarbonate (27.1 mg, 0.322 mmol)were filled in a vial, and then a physiological saline solution (0.902mL) was gradually added. These compounds dissolved while generatingcarbon dioxide gas and gave a clear solution. This solution was dilutedwith a physiological saline solution to 2.0 mL, whereby a medicationsolution having a concentration of 50 mg/mL was prepared.

Experimental Example 3

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol), sodium carbonate (42.7 mg, 0.403mmol) and sodium hydrosulfite (29.2 mg, 0.168 mmol) were filled in avial, and then a physiological saline solution (5 mL) was added to givea clear solution.

Experimental Example 4

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol), sodium carbonate (42.7 mg, 0.403mmol) and sodium sulfite (21.2 mg, 0.168 mmol) were filled in a vial,and then a physiological saline solution (5 mL) was added to give aclear solution.

Experimental Example 5

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol), sodium carbonate (42.7 mg, 0.403mmol) and sodium sulfite (0.42 mg, 0.003 mmol) were filled in a vial,and then a physiological saline solution (5 mL) was added to give aclear solution.

Experimental Example 6

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol) and sodium carbonate (42.8 mg, 0.403mmol) were filled in a vial, and then a 5% glucose solution (5 mL)containing sodium sulfite (1.25 mg) was added to give a clear solution.

Experimental Example 7

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol) and sodium carbonate (42.8 mg, 0.403mmol) were filled in a vial, and then a 5% glucose solution (5 mL)containing sodium hydrogen sulfite (1.25 mg) was added to give a clearsolution.

Experimental Example 8

3-[4-(l-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mmol) and sodium carbonate (42.8 mg, 0.403mmol) were filled in a vial, and then a 5% glucose solution (5 mL)containing sodium pyrosulfite (1.25 mg) was added to give a clearsolution.

Experimental Example 9

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (250 mg, 0.336 mol) and sodium carbonate (42.8 mg, 0.403mmol) were filled in a vial, and than a 5% glucose solution (5 mL)containing L-cysteine (1.25 mg) was added to give a clear solution.

Experimental Example 10

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (567 mg, 0.76 mmol), L-arginine (381.6 mg, 2.19 mmol) andsodium sulfite (4.6 mg, 0.036 mmol) were filled in a vial, and then aphysiological saline solution (50 mL) was added to give a clearsolution.

Experimental Example 11

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (567 mg, 0.76 mmol), L-arginine (381.6 mg, 2.19 mmol) andsodium sulfite (18.4 mg, 0.15 mmol) were filled in a vial, and then aphysiological saline solution (50 mL) was added to give a clearsolution.

Experimental Example 12

L-Arginine (81.4 mg, 3.2 equivalents) and sodium sulfite (1.8 mg, 0.1equivalent) were added to3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (113.4 mg), and filled in a 13P vial. The space wassubstituted by low humidity air to give a pharmaceutical preparation.

The obtained formulation was dissolved in a physiological salinesolution (2 mL), which remained clear for 24 hr.

The obtained pharmaceutical preparation was subjected to a stabilitytest. The formulation was stable as shown by the results in Table 1.

TABLE 1 Remaining Dissolution Preservation conditions ratio state* 60°C. × 2 weeks 97.7% clear 60° C. × 4 weeks 96.4% clear 40° C./75% RH × 1month 99.6% clear 40° C./75% RH × 2 months 95.8% clear 25° C. × 1 month100.4% clear 25° C. × 2 months 98.0% clear RH: relative humidity *:Dissolution state for 24 hr after dissolution in a physiological salinesolution (2 mL).

Experimental Example 13

L-Arginine (763.3 mg, 3.0 equivalents) and sodium sulfite (18.4 mg, 0.1equivalent) were added to3-[4-(1-methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (1.135 g) and filled in a 35K vial. The space wassubstituted by low humidity air to give a pharmaceutical preparation.

The obtained formulation was dissolved in a physiological salinesolution (10 mL), which remained clear for 24 hr.

Experimental Example 14

3-[4-(1-Methyl-4-pyridinio)-2-thiazolylthio]-7β-[2-(5-phosphonoamino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-cephem-4-carboxylate.aceticacid solvate (1.135 g) was filled in a 35K vial. The space wassubstituted by low humidity air and sealed with a rubber cap to give apharmaceutical preparation.

As sole use solvent, L-arginine (763.3 mg, 3.0 equivalents) and sodiumsulfite (18.4 mg, 0.1 equivalent) were dissolved in 10 mL of distilledwater and filled in an ampoule (10P). The space was substituted bynitrogen and the ampoule was melt-sealed.

The above-mentioned formulation was dissolved with the sole use solvent.The solution remained clear for 24 hr after dissolution, showing thesame level of quality of injection as in Example 14.

The L-arginine content and sodium sulfite content after autoclaving thesole use solvent at 121° C.×20 min were measured by potentiometer andion chromatography, respectively. As shown in Table 2, the sole usesolvent did not show degradation in quality even after autoclaving.

TABLE 2 Autoclave Sodium sulfite Dissolution treatment L-Argininecontent state applied 100.2% 99.8% clear none  99.0% 99.0% clear

INDUSTRIAL APPLICABILITY

The compound (particularly crystal) of the present invention shows highsolid stability and can be used as an antibacterial agent (particularlyanti-MRSA agent) having superior quality, such as possible long-termstable preservation and the like.

This application is based on patent application Nos. 247966/2000 and354959/2000 filed in Japan, the contents of which are herebyincorporated by reference.

1. A compound of the formula:

wherein X is CH₃COOH or CH₃CH₂COOH, and n is 0 to
 5. 2. The compound ofclaim 1, which is in the form of a crystal.
 3. The compound of claim 1,wherein n is
 1. 4. The compound of claim 1, wherein X is CH₃COOH.
 5. Thecompound of claim 4, having peaks near diffraction angles of 16.32,19.06, 19.90, 20.98 and 23.24° in powder X-ray diffraction (CuKα). 6.The compound of claim 4, having peaks near diffration angles of 11.82,17.16, 17.80, 19.32, 20.00, 21.20, 21.78, 22.94, 24.10 and 27.02° inpowder X-ray diffraction (CuKα).
 7. The compound of claim 1, wherein Xis CH₃CH₂COOH.
 8. The compound of claim 7, having peaks near diffractionangles of 16.30, 18.84, 19.70, 21.80 and 23.18° in powder X-raydiffraction (CuKα).
 9. A pharmaceutical composition, comprising thecompound of claim 1 and a pharmaceutically acceptable carrier, diluentor exipient.
 10. A production method of a crystal of a compoundrepresented by the formula:

wherein X is CH₃COOH or CH₃CH₂COOH, and n is 0 to 5, which comprisesmixing [i] a compound represented by the formula:

[ii] CH₃COOH or CH₃CH₂COOH and [iii] water, dissolving them and allowingcrystallization to take place.
 11. The production method of claim 10,wherein the proportion (volume ratio) to be used of CH₃COOH orCH₃CH₂COOH:water is 1:0.1-10.
 12. A crystal obtained by mixing [i] acompound represented by the formula:

[iii] CH₃COOH or CH₃CH₂COOH and [iii] water, dissolving them andallowing crystallization to take place.
 13. A disodium salt of acompound represented by the formula:


14. The disodium salt of claim 13, which is in the form of a crystal.15. The disodium salt of claim 13, having peaks near diffraction anglesof 17.02, 18.94, 22.86, 23.36 and 26.48° in powder X-ray diffraction(CuKα).
 16. The compound of claim 2, wherein X is CH₃COOH.
 17. Thecompound of claim 2, wherein X is CH₃CH₂COOH.
 18. A pharmaceuticalcomposition, comprising the compound of claim 2 and a pharmaceuticallyacceptable carrier, diluent or excipient.
 19. The disodium salt of claim14, having peaks near diffraction angles of 17.02, 18.94, 22.86, 23.36and 26.48° in powder X-ray diffraction (CuKα).
 20. A method for making acrystal of a compound of formula (I):

wherein X is CH₃COOH or CH₃CH₂COOH, and n is 0 to 5, comprisingcrystallizing a compound of formula (Ia):

from CH₃COOH or CH₃CH₂COOH and water.
 21. A crystal obtained bycrystallizing a compound of formula (Ia):

from CH₃COOH or CH₃CH₂COOH and water.